PROJECT SUMMARY/ABSTRACT Vesicular glutamate transporters (VGLUTs) package the major excitatory neurotransmitter glutamate into synaptic vesicles, thus play a critical role in quantal release and neurotransmission. Despite decades of research, the mechanism of glutamate transport by the VGLUTs and its regulation under different ionic and pH conditions during exocytosis and endocytosis remains poorly understood. The lack of molecular level understanding of the structure and function of VGLUTs severely hinders our ability to understand their role in normal brain function, as well as in many psychiatric and neurological conditions where malfunction of VGLUTs has been implicated. This proposal aims to bridge this gap by providing a molecular blue print of a mammalian VGLUT protein with atomic details. Specifically, the candidate Dr. Fei Li will determine high-resolution structures of the rat VGLUT1 (rVGLUT1) by cryo electronmicrosocpy (cryo-EM) (Aim 1, mentored) and characterize its function by electrophysiology (Aim 2, mentored). Dr. Fei Li will further determine the structure of the native synaptic vesicle by cryo electrontomography (cryo-ET) (Aim 3A, mentored) and to reveal the higher order organization and interactions of synaptic vesicle membrane proteins (Aim 3B, independent). The goal of this K99/R00 Pathway to Independence Award proposal is to enhance Dr. Li?s knowledge in neuroscience and to provide opportunity of additional training in several key techniques that are critical to launching her independent research career focusing on the structure and function of neuronal membrane proteins in a leading research institution. The proposed aims allow Dr. Li to explore new biological questions that she can continue to investigate in her independent career while providing opportunities for training at the same time. Dr. Li has a strong interest in membrane protein structure and function in the context of brain physiology, and a strong background in membrane protein and X-ray crystallography. To guide the proposed project and to enhance her career prospects, Dr. Li has selected three leading scientists in, respectively, membrane proteins (Dr. Robert Stroud), electron microscopy (Dr. Yifan Cheng), as well as electrophysiology and neuroscience (Dr. Robert Edwards) as her co-mentors. Training through this project will allow Dr. Li to build a highly versatile and integrative skill set extending her background in membrane proteins and X-ray crystallography to the state-of-the-art cryo-EM and cryo-ET technology. Skills in electrophysiology will provide her with the tools required for functional analysis. Together, the research project and training program proposed in this award will play a critical role in preparing Dr. Li for a successful independent research career.